Interferons (IFNs) are a group of proteins produced by the cells of most vertebrates and characterized by their ability to inhibit the growth of virus cells exposed to these proteins.
Interferons are species specific, that is active mainly on cells from the same animal species from which they have been obtained. Thus human cells will be sensitive to human IFNs, but not to most animal IFNs. Human IFNs are classified in three groups: IFN .alpha. and .beta. (also called type I IFNs) are produced by many cells of the body in response to viral inducers; in contrast, IFN-.gamma. (also called type II IFN) is produced by certain T-lymphocytes in response to antigens or mitogens and its production is part of the immune response. IFN-.gamma. differs from type I IFNs in its structure and in its functions. In particular, IFN-.gamma. induces the synthesis of histocompatibility antigens (HLA class I and class II) which assemble on the surface cells and mediate the immune response of lymphocytes, at concentrations which are much lower than those needed to inhibit virus proliferation (Wallach et al, Nature 299, 833-836, 1982). This property of IFN-.gamma. probably contributes in a most important way to the recognition and destruction of virus-infected cells, and of other modified cells such as cancer cells, by the immune system. It can be hoped therefore that IFN-.gamma. will prove efficient in clinical trials to stimulate the immune system to destroy foci of viral infection and tumors.
A major obstacle to testing the clinical efficacy of IFN-.gamma. is the small amount of IFN-.gamma.. This was prepared from fresh human peripheral mononuclear cells stimulated by a combination of phorbol myristate and lectins. Such methods can yield only 10-20,000 units IFN-.gamma. per ml of culture. Isolation of complementary DNA clones of the human IFN-.gamma. mRNA sequence allowed one researcher to produce the protein in E. coli with much higher yields (Gray et al, Nature 295, 503-508, 1982). The bacterial IFN-.gamma. differs, however, from the natural molecule by being unglycosylated and carrying 3-4 additional N-terminal amino acids.
However, the natural form of interferon gamma may be obtained from transformed mammalian cells. Haynes and Weissman, Nucleic Acids Res. 11: 687-706, (1983) constructed a plasmid bearing the dihydrofolate reductase (DHFR) gene under the control of the adenovirus major late promoter and a human gamma interferon cDNA gene under the control of the SV40 early promoter (derived from PKCR). Chinese hamster ovary (CHO) cells transformed by this plasmid expressed IFN-.gamma. at a rate of 20,000-100,000 units.ml.sup.-1.day.sup.-1, in clones selected after exposure to 0.2 or 1.0 micromoles methotrexate. Unamplified CHO cells produced IFN-.gamma. at a rate of 10-300 units.ml.sup.-1.day.sup.-1.
Scahill, et al., Proc Nat. Acad. Sci. USA, 80: 4654-58 (1983) cotransformed CHO cells with a first plasmid encoding the selectable marker DHFR and a second bearing a human gamma interferon cDNA gene under the control of the SV40 early promoter (derived from PSV2). CHO clones selected in methotrexate produced 50,000 units per ml. of culture medium.
Since the DHFR gene confers resistance to high concentratons of methotrexate, the cotransformed interferon gene is amplified by selection of transformed cells resistant to methotrexate, according to the method of Alt, et al., J. Biol. Chem., 253: 1357-70 (1978). See also, Bostock and Tyler-Smith, J. Mol. Biol., 153: 219-236 (1981); Nunberg, et al, PNAS (USA), 75: 5553-56 (1978).
The genomic human interferon gamma gene has been isolated and sequenced, and is thus known to include large intervening sequences (introns). Gray and Goeddel, Nature, 298: 859-863 (1982). Even though it is recognized that introns may be important in the formation of stable mRNAs, see Prochownik and Orkin, J. Biol. Chem, 259: 15386-15392 (Dec. 25, 1984), or may possess enhancer sequences, see Grilles, et al., Cell, 33: 717-728 (1983), interferon gamma cDNA has been utilized in the construction of eukaryotic interferon expression vectors. Goeddel and Gray, EP Appl. 77,670 (Apr. 27, 1983 publication).
While promoters are control elements which are effective only when positioned immediately 5' of the gene to be controlled, enhancers act relatively independently of their distance and orientation with respect to the coding region of the gene. Gruss, DNA 3: 1-5 (1984); Marx, Science, 221: 735-737 (Aug. 19, 1983). An enhancer region has been identified in the long terminal repeat (LTR) of the Harvey Murine Sarcoma virus. Kriegler .differential. Botchan, Mol. and Cell. Biol. 3: 325-339 (1983). Enhancer sequences may enhance the expression of heterologous genes, but their activity is believed to be host-specific and their mechanism of action is uncertain. Gruss, supra.
Berg, et al., Mol. and Cell. Biol., 3: 1246-54 (1983) have examined the effect of the SV40 72 bp repeat and the Murine Harvey Sarcoma virus 73 bp repeat enhancer sequences on the mouse beta globin promoter's control over the transcription of the E. coli galactokinase gene. They found that the Sv40 enhancer was stronger in CV-1 (primate) cells, while the reverse was true in L (mouse) cells. They also found that the effects of the two enhancers when present on the same DNA was additive.Luciw, et al., Cell, 33: 705-716 (1983) reported "provisional evidence that enhancer-promoter combinations functional in one set of experimental conditions may not work in another" (microinjection v. transfection). Indeed, the findings of Emerman and Temin, Cell, 39: 459-467 (1984), suggest that an enhancer may inhibit expression of an exogenuous gene under some circumstances. Thus, success in enhancing expression of one exogenous does not guarantee success in expressing another.